Hepatic fibrosis is the outcome of many chronic liver diseases, including cholestatic liver injury (primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), secondary biliary cirrhosis (SBC)) and hepatotoxic injury (hepatitis B virus (HBV), hepatitis C virus (HCV), alcoholic liver disease and non-alcoholic steatohepatitis (NASH)) (1). Hepatic fibrosis results from deregulation of wound healing with accumulation of extracellular matrix (ECM), including collagen type I, leading to scar formation. Several early events play an important role in the pathogenesis of liver fibrosis, including damage to hepatocytes, release of TGF-β1, the major fibrogenic cytokine, recruitment of inflammatory cells, induction of reactive oxygen species (ROS), and activation of ECM producing myofibroblasts, which are not present in the normal liver (1, 37). Activation of myofibroblasts is a critical step in liver fibrosis, and, therefore, myofibroblasts represent a primary target for antifibrotic therapy.
Fibrogenic myofibroblasts are cells responsible for collagen production and making the tissues fibrotic, the process associated with tissue destruction in organs capable of developing fibrosis, such as heart, lung, liver, kidney and skin diseases. Chronic liver injury of any etiology produces fibrosis as a result of deregulation of the normal healing process with massive accumulation of extracellular matrix (ECM), including type I collagen (ColI)(1). Myofibroblasts are ColI+ α-smooth muscle actin (α-SMA)+ cells that produce the ECM scar in fibrosis. One of the most important concepts in clinical and experimental liver fibrosis is reversibility. Removal of the etiological source of the chronic injury in patients (e.g. HBV, HCV, biliary obstruction, or alcohol) and in rodents (CCl4 or bile duct ligation) produces regression of liver fibrosis and is associated with decreased cytokine and ECM production, increased collagenase activity, and the disappearance of myofibroblasts (1, 2). During regression of fibrosis, some myofibroblasts undergo senescence (3) and apoptosis (2). However, the number of apoptotic myofibroblasts and the fate of the remaining myofibroblasts in the recovering liver is unknown.
The cells of origin of hepatic myofibroblasts are unresolved, and perhaps the fibrosis induced by different types of liver injury results from different fibrogenic cells. Hepatic myofibroblasts may originate from bone marrow-derived mesenchymal cells and fibrocytes, but only a small contribution of bone marrow (BM)-derived cells to the myofibroblast population has been detected in experimental liver fibrosis (48, 57-58). Another potential source of myofibroblast is epithelial-to-mesenchymal transition (EMT), in which epithelial cells acquire a mesenchymal phenotype and may give rise to fully differentiated myofibroblasts. However, recent cell fate mapping studies have failed to detect any hepatic myofibroblasts originating from hepatocytes, cholangiocytes, or epithelial progenitor cells (1, 59-63). Thus, the major sources of myofibroblasts in liver fibrosis are the endogenous liver mesenchymal cells, which consist of portal fibroblasts and hepatic stellate cells.
Hepatic stellate cells (HSCs), the liver pericytes that store retinoids, are a major source of myofibroblasts in hepatotoxic liver fibrosis (4). Liver injury results in activation of quiescent HSCs (qHSCs), which proliferate and undergo phonotypical and morphological changes characteristic of myofibroblasts. Removal of the injurious agent results in the clearance of activated HSCs (aHSCs) by the cytotoxic action of natural killer cells (1), and is linked to upregulation of ligands of NK cell receptor NKG2D, MICA and ULBP2, in senescent aHSCs (3). Although never demonstrated in vivo, studies in culture suggest that aHSCs can revert to a more quiescent phenotype (5), characterized by expression of adipogenic genes and loss of fibrogenic gene expression (5).
Portal fibroblasts normally comprise a small population of the fibroblastic cells that surround the portal vein to maintain integrity of portal tract. They were first described as “mesenchymal cells not related to sinusoids”, and since then were called “periductular fibroblasts” or “portal/periportal mesenchymal cells” (64) and implicated by association in the pathogenesis of cholestatic liver injury. In response to chronic injury, portal fibroblasts may proliferate, differentiate into α-SMA-expressing myofibroblasts, and synthesize extracellular matrix (64) (65-67).
The contribution of portal fibroblasts (PFs) to liver fibrosis of different etiologies is not well understood, mainly because of difficulties in isolating PFs and myofibroblasts. The most widely used method of PF isolation from rats is based on liver perfusion with enzymatic digestion followed by size selection (68). Cell outgrowth from dissected bile segments is still used to isolate mouse PFs, and after 10-14 days in culture PFs undergo progressive myofibroblastic activation (69). The disadvantage of this technique is that it requires multiple passaging and prolong culturing (64). A more physiological method of PF culturing in a precision-cut liver slice (PCLS) is designed to maintain cell-cell and cell-matrix interactions and mimic natural microenvironment of PFs, but does not enable the study of purified PFs (70). Therefore, only a few markers of PFs are available to identify PFs in the myofibroblast population, including gremlin, Thy1, fibulin 2, IL-6, elastin, the ecto-AT-Pase nucleoside triphosphate diphosphohydrolase-2 (NTPD2), and coffilin 1. In addition, the lack of desmin, cytoglobin, α2-macroglobulin, neural proteins (glial fibrillar acidic protein (GFAP), p75, synaptophysin), and lipid droplets distinguishes PFs from HSCs (1, 56, 71-74).